Methods for treatment of fibrotic diseases

ABSTRACT

Methods for treatment of fibrotic diseases using compounds of formula Iwherein RA is hydrogen,R7 and R8 are independently selected from H and SO2NR3R4,one of R7 and R8 is hydrogen, andR1, R2, R3, and R4 are each independently selected from H, alkyl, heteroalkyl, cycloalkyl, arylcycloalkyl, aryl, heteroaryl, heterocycloalkyl, and each of NR1R2 and NR3R4 can independently combine to form a heterocycloalkyl, and wherein said alkyl, heteroalkyl, cycloalkyl, arylcycloalkyl, aryl, heteroaryl, or heterocycloalkyl may be optionally substituted,or a pharmaceutically acceptable salt, ester, amide, stereoisomer, geometric isomer or prodrug thereof.

FIELD OF THE INVENTION

The present invention relates to the field of treatment of fibroticdiseases, including pulmonary fibrosis, Dupuytren's contracture,scleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis, keloids,chronic kidney disease, chronic graft rejection, and otherscarring/wound healing abnormalities, post-operative adhesions, reactivefibrosis, desmoid tumors and other conditions.

BACKGROUND OF THE INVENTION

Fibrotic diseases, including pulmonary fibrosis, Dupuytren'scontracture, scleroderma, systemic sclerosis, scleroderma-likedisorders, sine scleroderma, liver cirrhosis, interstitial pulmonaryfibrosis, keloids, chronic kidney disease, chronic graft rejection, andother scarring/wound healing abnormalities, post-operative adhesions,reactive fibrosis, and desmoid tumors are important conditions whichoften cause morbidity and mortality and can affect all tissues and organsystems.

Pulmonary fibrosis is a condition in which the lung tissue becomesthickened, stiff, and scarred. While the cause of the fibrosis(scarring) can be sometimes determined, often the etiology of thiscondition remains unknown. When there is no known cause for thedevelopment of pulmonary fibrosis (and certain radiographic and/orpathologic criteria for pulmonary fibrosis are met), the disease iscalled idiopathic pulmonary fibrosis (IPF).

IPF has no specific demographic profile, and may be found in both urbanand rural environments. Risk factors of IPF include smoking and certaingenetic factors. IPF affects more men than women and usually occursbetween the ages of 50 and 70.

Dupuytren's contracture, which is alternatively known as palmarfibromatosis (or “Dupuytren's disease”), is a disease associated withthe build-up of extracellular matrix materials such as collagen on theconnective tissue of the hand (the palmar fascia), causing it to thickenand shorten with the physical effect of causing the fingers to curl,most commonly the ring finger and little finger. Dupuytren's contractureis manifested through progressive flexion contracture of the digits ofthe hand, resulting in significantly compromised function. It affectsboth males and females, but the incidence is higher in males.

The causes of Dupuytren's disease are not well understood and underlyingdisease is not currently curable.

Accordingly, there is a need for novel methods of treating fibroticdiseases.

SUMMARY OF THE INVENTION

The present invention is directed to the observation the WNT/betacatenin signaling is an important mediator of fibrotic diseases and thatinhibition of this signaling pathway ameliorates fibrosis and fibroticdisease states. Inhibitors of the Wnt/beta-catenin signaling pathway canbe used for the treatment and/or prevention of fibrotic diseases,including but not limited to pulmonary fibrosis, Dupuytren'scontracture, scleroderma, systemic sclerosis, scleroderma-likedisorders, sine scleroderma, liver cirrhosis, interstitial pulmonaryfibrosis, keloids, chronic kidney disease, chronic graft rejection, andother scarring/wound healing abnormalities, post-operative adhesions,reactive fibrosis, and desmoid tumors.

The invention includes all possible methods of administration, includingintravenous, parenteral, oral, inhalation (including aerosolizeddelivery), buccal, intranasal, rectal, intra-lesional, intraperitoneal,intradermal, transdermal, subcutaneous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreous injection, andtopical application.

In one embodiment, the invention is directed to aerosolized delivery ofsuch compounds, in particular for treating pulmonary conditions. Inanother embodiment, the invention provides intravenous injection fortreating Dupuytren's contracture. In yet another embodiment, theinvention provides topical application for treatment of keloids.

Accordingly, the present invention provides methods for treating and/orpreventing fibrotic diseases comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of formulaI

wherein R_(A) is hydrogen,

R₇ and R₈ are independently selected from H and SO₂NR₃R₄, one of R₇ andR₈ is hydrogen, and

R₁, R₂, R₃, and R₄ are each independently selected from H, alkyl,heteroalkyl, cycloalkyl, arylcycloalkyl, aryl, heteroaryl,heterocycloalkyl, and each of NR₁R₂ and NR₃R₄ can independently combineto form a heterocycloalkyl, and wherein said alkyl, heteroalkyl,cycloalkyl, arylcycloalkyl, aryl, heteroaryl, or heterocycloalkyl may beoptionally substituted,

or a pharmaceutically acceptable salt, ester, amide, stereoisomer,geometric isomer or prodrug thereof.

U.S. Pat. No. 8,129,519 describes methods of making the compounds of theinvention. In one embodiment, the invention contemplates the use of anyof the compounds of the U.S. Pat. No. 8,129,519 for treating and/orpreventing fibrotic diseases according to the present invention.

In one preferred embodiment, NR₁R₂ and NR₃R₄ are independently 6- to15-membered heterocycloalkyl containing one nitrogen in the ring.

In a preferred embodiment, the compound of formula I has the followingstructure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer,geometric isomer or prodrug thereof. This compound is also known asBC-2059 or Tegavivint. U.S. Pat. No. 8,129,519 describes methods ofmaking this compound.

In one embodiment, the fibrotic disease is selected from the groupconsisting of pulmonary fibrosis, Dupuytren's contracture, scleroderma,systemic sclerosis, scleroderma-like disorders, sine scleroderma, livercirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidneydisease, chronic graft rejection, and other scarring/wound healingabnormalities, post-operative adhesions, reactive fibrosis.

In a preferred embodiment, the disorder is pulmonary fibrosis.

In another preferred embodiment, the disorder is Dupuytren'scontracture.

In yet another preferred embodiment, the disorder is keloids.

In one embodiment, the invention provides a method of treating pulmonaryfibrosis comprising a systemic administration or an aerosolized deliveryof Tegavivint to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a graph of cell growth inhibition of primary palmar fasciafibroblasts derived from the fibrotic palmar fascia cells of a patientwith Dupuytren's Disease (DD) (DD249 cells) growth rate.

FIG. 1B shows a chart of cell growth inhibition of DD249 25 pMabsorbance.

FIG. 1C shows a chart of cell growth inhibition of DD249 50 pMabsorbance.

FIG. 1D shows a graph of cell growth inhibition of syngeneic primarypalmar fascia fibroblasts derived from visibly non-fibrotic PalmarFascia (PF) from the patients with Dupuytren's disease (PF249 cells)growth rate.

FIG. 1E shows a chart of cell growth inhibition of PF249 25 pMabsorbance.

FIG. 1F shows a chart of cell growth inhibition of PF249 50 pMabsorbance.

FIG. 2A shows cell growth inhibition of primary palmar fasciafibroblasts derived from the fibrotic palmar fascia cells of a patientwith Dupuytren's Disease (DD) (DD77 cells).

FIG. 2B shows cell growth inhibition of syngeneic primary palmar fasciafibroblasts derived from visibly non-fibrotic Palmar Fascia (PF) from apatient with Dupuytren's disease (PF77 cells).

FIG. 3A shows gene expression analysis of CTNNB1 gene expression insyngeneic DD cells cultured from the explant tissues from one patientwith Dupuytren's disease (DD180 cells); syngeneic PF cells cultured fromthe explant tissues from this patient with Dupuytren's disease (PF180cells); and syngeneic DD cells cultured from the explant tissues fromone patient with no history of Dupuytren's disease (CT38 cells).

FIG. 3B shows gene expression analysis of EGR1 gene expression in DD180,PF180 and CT38 cells.

FIG. 3C shows gene expression analysis of NRG1 gene expression in DD180,PF180 and CT38 cells.

FIG. 3D shows gene expression analysis of WISP1 gene expression inDD180, PF180 and CT38 cells.

FIG. 4A shows Fibroblast-populated collagen lattice (FPCL) contractionassays of DD180 cells at 4.5 hours after lattice release.

FIG. 4B shows FPCL contraction assays of DD180 cells at 24 hours afterlattice release.

FIG. 5A shows pulmonary compliance analysis in bleomycin-inducedpulmonary fibrosis mouse model with or without intravenous Tegavivinttreatment.

FIG. 5B shows Sircol collagen assay results in bleomycin-inducedpulmonary fibrosis mouse model with or without intravenous Tegavivinttreatment.

FIG. 6A shows pulmonary compliance analysis in bleomycin-inducedpulmonary fibrosis mouse model with or without intranasal Tegavivinttreatment.

FIG. 6B shows Sircol collagen assay results in bleomycin-inducedpulmonary fibrosis mouse model with or without intranasal Tegavivinttreatment.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following definitions are used, unless otherwise described.

The term “subject” includes mammals, including humans. The terms“patient” and “subject” are used interchangeably.

The term “therapeutically effective amount” means the amount of acompound that, when administered to a subject for treating a disease ordisorder, is sufficient to affect the disease or disorder. The“therapeutically effective amount” can vary depending on the variety offactors, including the compound, the disorder being treated and theseverity of the disorder; activity of the specific compound employed;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the compound at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

In one embodiment, the terms “treating” or “treatment” refer toameliorating the disease or disorder (i.e., arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment, “treating” or “treatment” refers toameliorating at least one physical parameter, which may not bediscernible by the subject. In yet another embodiment, “treating” or“treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers to delayingthe onset of the disease or disorder, or even preventing the same.

The phrase “pharmaceutically acceptable salt” means those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well-known in the art. For example, S. M. Berge etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66: 1 et seq.

Pharmaceutically acceptable salts include, but are not limited to, acidaddition salts. For example, the nitrogen atoms may form salts withacids. Representative acid addition salts include, but are not limitedto acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate,glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, thebasic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which canbe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulfuric acid and phosphoric acid and such organic acids as oxalic acid,maleic acid, succinic acid and citric acid.

Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylammonium,dimethylammonium, trimethylammonium, triethylammonium, diethylammonium,and ethylammonium among others. Other representative organic aminesuseful for the formation of base addition salts include ethylenediamine,ethanolamine, diethanolamine, piperidine, piperazine and the like.

Compounds useful for the purposes of the invention can contain one ormore double bonds and, thus, potentially give rise to cis/trans (E/Z)isomers, as well as other conformational isomers. Unless stated to thecontrary, the invention includes all such possible isomers, as well asmixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and/or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture.

Many of the compounds described herein can have one or more chiralcenters and therefore can exist in different enantiomeric forms. Ifdesired, a chiral carbon can be designated with an asterisk (*). Whenbonds to the chiral carbon are depicted as straight lines in thedisclosed formulas, it is understood that both the (R) and (S)configurations of the chiral carbon, and hence both enantiomers andmixtures thereof, are embraced within the formula. As is used in theart, when it is desired to specify the absolute configuration about achiral carbon, one of the bonds to the chiral carbon can be depicted asa wedge (bonds to atoms above the plane) and the other can be depictedas a series or wedge of short parallel lines is (bonds to atoms belowthe plane). The Cahn-Inglod-Prelog system can be used to assign the (R)or (S) configuration to a chiral carbon.

Compounds useful for the purposes of the invention may comprise atoms inboth their natural isotopic abundance and in non-natural abundance. Thedisclosed compounds can be isotopically-labelled orisotopically-substituted compounds identical to those described, but forthe fact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numbertypically found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

Compounds further comprise prodrugs thereof, and pharmaceuticallyacceptable salts of said compounds or of said prodrugs which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this invention. Certain isotopically-labelled compounds ofthe present invention, for example those into which radioactive isotopessuch as ³H and ¹⁴C are incorporated, are useful in drug and/or substratetissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e.,¹⁴C, isotopes are particularly preferred for their ease of preparationand detectability. Further, substitution with heavier isotopes such asdeuterium, i.e., ²H, can afford certain therapeutic advantages resultingfrom greater metabolic stability, for example increased in vivohalf-life or reduced dosage requirements and, hence, may be preferred insome circumstances. Isotopically labelled compounds of the presentinvention and prodrugs thereof can generally be prepared by carrying outthe procedures below, by substituting a readily available isotopicallylabelled reagent for a non-isotopically labelled reagent.

The compounds useful for the purposes of the invention can be present asa solvate. In some cases, the solvent used to prepare the solvate is anaqueous solution, and the solvate is then often referred to as ahydrate. The compounds can be present as a hydrate, which can beobtained, for example, by crystallization from a solvent or from aqueoussolution. In this connection, one, two, three or any arbitrary number ofsolvate or water molecules can combine with the compounds according tothe invention to form solvates and hydrates. Unless stated to thecontrary, the invention includes all such possible solvates.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with anα-hydrogen can exist in an equilibrium of the keto form and the enolform.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain aspects,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

For example, a “C1-C3 alkyl” group can be selected from methyl, ethyl,n-propyl, i-propyl, and cyclopropyl, or from a subset thereof. Incertain aspects, the “C1-C3 alkyl” group can be optionally furthersubstituted. As a further example, a “C1-C4 alkyl” group can be selectedfrom methyl, ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl,s-butyl, t-butyl, and cyclobutyl, or from a subset thereof. In certainaspects, the “C1-C4 alkyl” group can be optionally further substituted.As a further example, a “C1-C6 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, and cyclohexane, or from a subset thereof. In certainaspects, the “C1-C6 alkyl” group can be optionally further substituted.As a further example, a “C1-C8 alkyl” group can be selected from methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, s-butyl,t-butyl, cyclobutyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, neopentyl,cyclopentyl, n-hexyl, i-hexyl, 3-methylpentane, 2,3-dimethylbutane,neohexane, cyclohexane, heptane, cycloheptane, octane, and cyclooctane,or from a subset thereof. In certain aspects, the “C1-C8 alkyl” groupcan be optionally further substituted. As a further example, a “C1-C12alkyl” group can be selected from methyl, ethyl, n-propyl, i-propyl,cyclopropyl, n-butyl, i-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl,i-pentyl, s-pentyl, t-pentyl, neopentyl, cyclopentyl, n-hexyl, i-hexyl,3-methylpentane, 2,3-dimethylbutane, neohexane, cyclohexane, heptane,cycloheptane, octane, cyclooctane, nonane, cyclononane, decane,cyclodecane, undecane, cycloundecane, dodecane, and cyclododecane, orfrom a subset thereof. In certain aspects, the “C1-C12 alkyl” group canbe optionally further substituted.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, nitrile,sulfonamide, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The terms “halogen,” “halide,” and “halo,” as used herein, refer to thehalogens fluorine, chlorine, bromine, and iodine. It is alsocontemplated that, in various aspects, halogen can be selected fromfluoro, chloro, bromo, and iodo. For example, halogen can be selectedfrom fluoro, chloro, and bromo. As a further example, halogen can beselected from fluoro and chloro. As a further example, halogen can beselected from chloro and bromo. As a further example, halogen can beselected from bromo and iodo. As a further example, halogen can beselected from chloro, bromo, and iodo. In one aspect, halogen can befluoro. In a further aspect, halogen can be chloro. In a still furtheraspect, halogen is bromo. In a yet further aspect, halogen is iodo.

It is also contemplated that, in certain aspects, pseudohalogens (e.g.triflate, mesylate, tosylate, brosylate, etc.) can be used in place ofhalogens. For example, in certain aspects, halogen can be replaced bypseudohalogen. As a further example, pseudohalogen can be selected fromtriflate, mesylate, tosylate, and brosylate. In one aspect,pseudohalogen is triflate. In a further aspect, pseudohalogen ismesylate. In a further aspect, pseudohalogen is tosylate. In a furtheraspect, pseudohalogen is brosylate.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds suitable for the purposes of theinvention may contain “optionally substituted” moieties. In general, theterm “substituted,” whether preceded by the term “optionally” or not,means that one or more hydrogens of the designated moiety are replacedwith a suitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. In is also contemplated that,in certain aspects, unless expressly indicated to the contrary,individual substituents can be further optionally substituted (i.e.,further substituted or unsubstituted).

Unless the present specification uses a different definition, all of thedefinitions and other disclosures of U.S. Pat. No. 8,129,519 areexpressly incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the observation the WNT/betacatenin signaling is an important mediator of fibrotic diseases and thatinhibition of this signaling pathway ameliorates fibrosis and fibroticdisease states. Inhibitors of the Wnt/beta-catenin signaling pathway canbe used for the treatment and/or prevention of fibrotic diseases,including but not limited to pulmonary fibrosis, Dupuytren'scontracture, scleroderma, systemic sclerosis, scleroderma-likedisorders, sine scleroderma, liver cirrhosis, interstitial pulmonaryfibrosis, keloids, chronic kidney disease, chronic graft rejection, andother scarring/wound healing abnormalities, post-operative adhesions,reactive fibrosis, and desmoid tumors.

Accordingly, the present invention provides methods for treating and/orpreventing fibrotic diseases comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of formulaI

wherein R_(A) is hydrogen,

R₇ and R₈ are independently selected from H and SO₂NR₃R₄, one of R₇ andR₈ is hydrogen, and

R₁, R₂, R₃, and R₄ are each independently selected from H, alkyl,heteroalkyl, cycloalkyl, arylcycloalkyl, aryl, heteroaryl,heterocycloalkyl, and each of NR₁R₂ and NR₃R₄ can independently combineto form a heterocycloalkyl, and wherein said alkyl, heteroalkyl,cycloalkyl, arylcycloalkyl, aryl, heteroaryl, or heterocycloalkyl may beoptionally substituted,

or a pharmaceutically acceptable salt, ester, amide, stereoisomer,geometric isomer or prodrug thereof.

In one preferred embodiment, NR₁R₂ and NR₃R₄ are independently 6- to15-membered heterocycloalkyl containing one nitrogen in the ring.

U.S. Pat. No. 8,129,519 describes methods of making the compounds of theinvention.

In a preferred embodiment, the compound of formula I has the followingstructure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof. The compound is also known as BC-2059 orTegavivint. U.S. Pat. No. 8,129,519 describes methods of making thiscompound.

In one embodiment, the fibrotic disease is selected from the groupconsisting of pulmonary fibrosis, Dupuytren's contracture, scleroderma,systemic sclerosis, scleroderma-like disorders, sine scleroderma, livercirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidneydisease, chronic graft rejection, and other scarring/wound healingabnormalities, post-operative adhesions, reactive fibrosis.

In a preferred embodiment, the disorder is pulmonary fibrosis.

In another preferred embodiment, the disorder is Dupuytren'scontracture.

In yet another preferred embodiment, the disorder is keloids.

BC-2059 was originally identified in a cell based screen for its abilityto inhibit the transcriptional activation of the WNT/beta cateninsignaling pathway. Characterization of this compound series led to thediscovery that members of this compound series were able to induce thedegradation of beta-catenin, interfere with the beta catenintranscriptional activation complex, and had characteristics of a nuclearreceptor signaling pathway modulator. BC-2059 was found to interact withTBL1 and prevent beta-catenin from associating with TBL1 and leads tobeta-catenin degradation.

This activity of BC-2059 was found to inhibit the beta catenin pathwayin cancer cells and cause those cells to undergo apoptosis.Specifically, cell lines derived from chromic myeloid leukemia (CML)patients and cell lines and primary cells derived frommyeloproliferative neoplasm (MPN) patients undergo apoptosis and growthinhibition in the presence of BC-2059. In addition, the activity ofBC-2059 is synergistic with compounds that affect therapeuticallyimportant signaling pathways in these diseases (such as Janus kinase 2(JAK2), break point cluster-Abelson (BCR-ABL), and Histone deacetylase(HDACs) inhibitors and can be used in combination with these agents toameliorate these diseases in individuals with the disease.

Thus, in some embodiments, the provided agents can be used incombination with other therapeutic agents, including but not limited totyrosine kinase inhibitor (including but not limited to nilotinib),histone deacetylase inhibitor (including, but not limited topanobinostat), other anti-cancer agents and other therapeutic agents.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the present invention can be employedin pure form or, where such forms exist, in pharmaceutically acceptablesalt, ester or prodrug form. Alternatively, the compound can beadministered as a pharmaceutical composition containing the compound ofinterest in combination with one or more pharmaceutically acceptableexcipients.

The total daily dose of the compounds of this invention administered toa human or lower animal may range from about 0.0001 to about 1000mg/kg/day. If desired, the effective daily dose can be divided intomultiple doses for purposes of administration, consequently, single dosecompositions may contain such amounts or submultiples thereof to make upthe daily dose.

For a clearer understanding of the invention, details are providedbelow. These are merely illustrations and are not to be understood aslimiting the scope of the invention in any way. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from thefollowing examples and foregoing description. Such modifications arealso intended to fall within the scope of the appended claims.

EXAMPLES Example 1 Effects of Tegavivint on Primary Cell Models ofFibrotic (Dupuytren's Disease) and Non-Fibrotic Palmar FasciaExperimental Conditions

Tegavivint (BC-2059) powder was reconstituted it in 100% dimethylsulfoxide (DMSO) to a 10_mM stock solution. The stock was diluted to 100nM in serum-free media (α minimal essential medium, αMEM), and furtherdiluted to 100 pM in serum-free αMEM immediately before the analyseswere performed. Serial dilutions from 100 pM down to 0.78 pM wereassessed in various assays. 0.001% DMSO in αMEM was used as the vehiclecontrol, and αMEM alone was used as an untreated control, in a subset ofanalyses.

Proliferation/viability analyses were performed in triplicate on primarypalmar fascia fibroblasts derived from the fibrotic palmar fascia (DDcells) of 2 patients with Dupuytren's disease (DD249, DD77) andsyngeneic primary palmar fascia fibroblasts derived from visiblynon-fibrotic palmar fascia (PF cells) from these patients (PF249, PF77).Cells were cultured on type-1 collagen coated 96 well trays for theseanalyses to better replicate substrate interactions in vivo. Twoindependent assays were used for these analyses: Alamar Blue assays foranalyses up to 72 hrs in cells derive from 1 patient, and Water SolubleTetrazolium-(WST-1) assays for analyses up to 24 hrs in cells derivedfrom the other patients. Significant effects of treatment onproliferation over time were detected by ANOVA repeated measures.

Gene expression analyses were assessed in triplicate on a Real-Time PCRABI Prism 7500. Total RNA samples were derived from syngeneic DD and PFcells cultured from the explant tissues from one patient withDupuytren's disease (DD180 and PF 180) and 1 patient with no history ofDupuytren's disease (CT38) as a normal allogeneic control. RNA qualitywas assessed on an Agilent 2100 Bioanalyzer and 2 μg of high qualitytotal RNA was reverse transcribed into cDNA first strand using theHigh-Capacity cDNA Archive Kit (Applied Biosystems) in accordance withthe manufacturer's instructions. TaqMan gene expression assays were usedto measure CTNNB1, EGR1, NRG1 and WISP1 mRNA levels after 48 hrs oftreatment in cells cultured on type-1 collagen coated 6 well trays. TheΔΔCT method was used after confirmation of parallel PCR amplificationefficiencies of target and housekeeping genes PCR reactions were carriedout under the following conditions: Initial denaturation at 95° C. for 5min followed by cycles of denaturation (95° C. for 15 sec), primerannealing (60° C. for 1 min) and transcript extension (50° C. for 2 min)for 40 cycles.

Fibroblast populated collagen lattice assays were performed on primarypalmar fascia fibroblasts derived from the fibrotic palmar fascia of onepatient with Dupuytren's disease (DD180) using the protocol described inRaykha, C., Crawford, J., Gan, B. S., Fu, P., Bach, L. A., and O'Gorman,D. B. (2013) IGF-II and IGFBP-6 regulate cellular contractility andproliferation in Dupuytren's disease. Biochimica et biophysica acta.1832, 1511-9.

Experimental Results

Based on the results of both proliferation/viability assays utilized andvisual inspection of the cells in culture, BC-2059 was cytotoxic forboth fibrosis-derived fibroblasts and syngeneic fibroblasts derived fromvisibly unaffected palmar fascia at concentrations >100 pM. Consistentand statistically significant differences in sensitivity to BC-2059 wereevident in the proliferation/viability assays of the DD and PF cells(N=2 patients) that we assessed. As shown in FIGS. 1A through 1C, DD249cells were viable and able to proliferate to significantly greater cellnumbers in 25 pM BC-2059 over 48 and 72 hrs relative to cells treatedfor 24 hrs, but were unable to proliferate in 50 pM BC-2059 over 48 and72 hrs relative to cells treated for 24 hrs.

In contrast, syngeneic PF249 cells proliferated to significantly greatercell numbers in both 25 pM and 50 pM BC-2059 over 48 and 72 hrs relativeto cells treated for 24 hrs. See, FIGS. 1D through 1F.

These findings were replicated using a different assay, WST-1, in cellsderived from a different patient, DD77 and PF 77, over 24 hrs, as shownin FIGS. 2A and 2B.

No discernible effects on the expression of genes that associate withnuclear 3-catenin in ChIP-seq analyses of DD cells were identified incells treated with 25_pM BC-2059.

CONCLUSION

Our preliminary analyses have identified a therapeutic window forconcentrations of BC-2059 at approximately 50 pM, where theproliferation of fibrosis-derived cells, but not syngeneic palmar fasciaderived cells, is inhibited. We did not identify any consistent effectson cell proliferation/viability, gene expression or contractility at ≤25pM BC-2059 in any of our analyses, providing further confirmation of atherapeutic window above 25 pM BC-2059.

BC-2059 inhibits nuclear localization of beta-catenin, and not itstranscription, however it was worthwhile to determine if there was anyevidence of a compensatory increase in CTNNB1 mRNA levels in BC-2059treated cells. As FIG. 3A shows, no evidence of a compensatory increasein CTNNB1 expression was detected in cells treated with ≤25_pM BC-2059.Our previous data indicate that beta-catenin associates withtranscription factors in the promoters of EGR1 and NRG1 in CT cells, butnot DD or PF cells, and that β-catenin associates with transcriptionfactors in the WISP1 promoter in DD cells, but not PF or CT cells. WhileEGR1 and NRG1 expression levels were highest in CT cells, and WISP1expression was highest in DD cells as expected, we did not see anyevidence of BC-2059-induced changes in EGR1, NRG1 or WISP1 expressionlevels in cells treated with ≤25_pM BC-2059. See, FIGS. 3B-3D.

To compensate for the genetic variability between cells derived fromdifferent patients, we recommend that additional, more detailed in vitroanalyses of BC-2059 be performed in the 25-100 pM range on cells derivedfrom the fibrotic and visibly non-fibrotic palmar fascia (i.e. DD and PFcells) of at least 6 additional individuals. We have calculated that 6patients/group (DD and PF) is sufficient to detect significance atp<0.05 with a power of 80%.

Example 2

Method of Treating Pulmonary Fibrosis with Tegavivint

In a pilot study, Tegavivint at 50 mg/kg was administered biw (twice aweek) via tail-vein injection to C57BL/6 wild-type mice. Bleomycin wasadministered to the mice intratracheally on day 0 to induce pulmonaryfibrosis, and Tegavivint or the vehicle (5% dextrose in water) wasadministered on days 6, 10, 14, 18 and 21.

The purpose of this experiment was to evaluate the effect ofsystemically-administered Tegavivint on bleomycin-induced pulmonaryfibrosis in vivo.

FIG. 5A demonstrates the effect on pulmonary compliance, which is afunctional measure of the lung's ability to stretch and expand.Pulmonary fibrosis is usually associated with decreased compliance, i.e.a stiff lung. As shown in FIG. 5A, animals treated with bleomycin only(second group) showed significantly decreased compliance compared withanimals treated with vehicle only (first group), indicating induction ofpulmonary fibrosis after bleomycin treatment; Tegavivint alone (thirdgroup) had no effect on compliance; and Tegavivint treatment afterbleomycin (fourth group) partially restored the compliance measurements.Asterisks denote statistical significance at ** p<0.01 and ***p<0.001.

FIG. 5B demonstrate the effect on total soluble collagen content in lungtissue using the Sircol assay, which is a quantitative measure ofpulmonary fibrosis, as the induction of fibrosis is associated increasednew collagen synthesis and elevated soluble collagen concentration. Asshown in FIG. 5B, animals treated with bleomycin only (second group)showed significantly increased soluble collagen concentration comparedwith animals treated with vehicle only (first group), indicatinginduction of pulmonary fibrosis after bleomycin treatment; Tegavivintalone (third group) had no effect; and Tegavivint treatment afterbleomycin (fourth group) completely restored the collagen level to thesame as vehicle-only controls. Asterisks denote statistical significanceat ***p<0.005.

This experiment has demonstrated that Tegavivint treatment effectivelyattenuated bleomycin-induced pulmonary fibrosis in vivo via systemicadministration.

In another pilot study, Tegavivint at 5 mg/kg was administered biw(twice a week) via intranasal delivery to C57BL/6 wild-type mice.Bleomycin was administered to the mice intratracheally on day 0 toinduce pulmonary fibrosis, and Tegavivint or the vehicle (5% dextrose inwater) was administered on days 6, 10, 14, 18 and 21.

The purpose of this experiment was to evaluate the effect oftopically-administered Tegavivint on bleomycin-induced pulmonaryfibrosis in vivo.

FIG. 6A demonstrates the effect of topical Tegavivint on pulmonarycompliance in the bleomycin model. As shown in FIG. 6A, animals treatedwith bleomycin only (second group) showed significantly decreasedpulmonary compliance compared with animals treated with vehicle only(first group), indicating induction of pulmonary fibrosis afterbleomycin treatment; Topical Tegavivint alone (third group) caused anincrease in compliance; and Tegavivint treatment after bleomycin (fourthgroup) showed a trend towards increased compliance compared to thebleomycin only (second) group, although more animals may be needed todemonstrate statistical significance. Asterisks denote statisticalsignificance at * p<0.05 and **p<0.01.

FIG. 6B demonstrates the effect of topical Tegavivint on solublecollagen content in the bleomycin model. As shown in FIG. 6B, animalstreated with bleomycin only (second group) showed significantlyincreased soluble collagen concentration compared with animals treatedwith vehicle only (first group), indicating induction of pulmonaryfibrosis after bleomycin treatment; Topical Tegavivint alone (thirdgroup) showed a slight increase in collagen, which could be due to amild inflammatory response of the lung tissue to the topical drug; andTegavivint treatment after bleomycin (fourth group) significantlydecreased the collagen level compared with the bleomycin-only (second)group. Asterisks denote statistical significance at **p<0.01,****p<0.0001.

This experiment has demonstrated that topically-administered Tegavivintalso effectively attenuated bleomycin-induced pulmonary fibrosis invivo.

Example 3 Nebulizing Delivery of Tegavivint Formulations

Tegavivint particles suspended in Poloxamer 188/sorbitol at aconcentration of 25 mg/mL were used. These formulations were applied tothe mice in the form of aerosols, through the method of whole bodyexposure. The mice were placed inside a plastic box. This box was sealedand connected by one of its sides to the outlet of the nebulizer device,and on the other side to a system of closed water. The whole procedurewas carried out inside the fume hood of the animal room.

For the first experiment, the nebulizer kit of SATER LABS was used. Thisdevice uses the jet system. The device was primed with 5 ml of the drug,i.e. 125 mg of tegavivint (BC2059), for each group of 5 mice, and thenthe device was connected to the power source for nebulization. Theenergy was supplied by a DeVilbiss compressor model 646, which allows5-7 pounds of pressure, and a flow of 6-8 liters per minute. For thesecond experiment, the device used was the Altera, ultrasonic nebulizer.

For the two experiments, 10 male bcat-Ex3 mice were used for each set.These mice were separated into 2 groups of 5 mice each. The first groupreceived the drug daily, for 5 consecutive days. The second groupreceived the drug only once (the fifth day). On day 5 all mice weresacrificed, lung harvested, and samples were stored at −30 degrees, intwo labeled nylon bags, each containing the 5 samples from each group.

Results:

Matrix/ Lung Lung Bleed Concentration Concentration Label Group IDAnimal ID time Analyte (ng/mL) (ng/g) Aerosol 1 Day #1-1 2 1 MouseBC2059 14200 56800 Oct. 10, 2017 lung Aerosol 1 Day #2-2 2 2 MouseBC2059 1300 5200 Oct. 10, 2017 lung Aerosol 1 Day #3-3 2 3 Mouse BC20594270 17100 Oct. 10, 2017 lung Aerosol 1 Day #4-4 2 4 Mouse BC2059 12605040 Oct. 10, 2017 lung Aerosol 1 Day #5-5 2 5 Mouse BC2059 4190 16800Oct. 10, 2017 lung Aerosol 5 Day #1-6 3 6 Mouse BC2059 4640 18600 Oct.10, 2017 lung Aerosol 5 Day #2-7 3 7 Mouse BC2059 1650 6600 Oct. 10,2017 lung Aerosol 5 Day #3-8 3 8 Mouse BC2059 3620 14500 Oct. 10, 2017lung Aerosol 5 Day #4-9 3 9 Mouse BC2059 3080 12300 Oct. 10, 2017 lungAerosol 5 Day #5-10 3 10 Mouse BC2059 3550 14200 Oct. 10, 2017 lungNebulizer 1 Day #1-11 4 11 Mouse BC2059 2840 11400 Oct. 10, 2017 lungNebulizer 1 Day #2-12 4 12 Mouse BC2059 2910 11600 Oct. 10, 2017 lungNebulizer 1 Day #3-13 4 13 Mouse BC2059 8660 34600 Oct. 10, 2017 lungNebulizer 1 Day #4-14 4 14 Mouse BC2059 3780 15100 Oct. 10, 2017 lungNebulizer 1 Day #5-15 4 15 Mouse BC2059 601 2400 Oct. 10, 2017 lungNebulizer 5 Day #1-16 5 16 Mouse BC2059 4770 19100 Oct. 10, 2017 lungNebulizer 5 Day #2-17 5 17 Mouse BC2059 4290 17200 Oct. 10, 2017 lungNebulizer 5 Day #3-18 5 18 Mouse BC2059 4690 18800 Oct. 10, 2017 lungNebulizer 5 Day #4-19 5 19 Mouse BC2059 3040 12200 Oct. 10, 2017 lungNebulizer 5 Day #5-20 5 20 Mouse BC2059 5730 22900 Oct. 10, 2017 lungLLOQ: 20.0 ng/g “Aerosol” refers to standard aerosol jet nebulizer(Sater Labs); “Nebulizer” refers to nebulizer ultrasonic eRapid machine(Altera) “1 day” refers to single-dose on Day 5; “5 day” refers to 5daily doses on Days 1-5.

Example 4 Efficacy of Nebulized Tegavivint in a Mouse Model ofIdiopathic Pulmonary Fibrosis

The purpose of this experiment was to investigate tegavivintnanosuspension in a mouse model of bleomycin-induced idiopathicpulmonary fibrosis (IPF). Test articles were as follows:

Tegavivint (BC2059) in a nano-milled suspension 25 mg/mL in 0.625%poloxamer 188 and 10% sorbitol. The compound was refrigerated at about4° C.

Nebulizing equipment was Altera ultrasonic eRapid machine nebulizer(model #678G1002).

Animals were 8-12 week old C57BL/6 male mice (Jackson Lab, Bar Harbor,Me.).

Experimental Procedure

Group # of mice Day 0 Day 5-21 1 4 IT PBS 50 μl 5 ml of Vehicle (0.625%poloxamer 188/10% sorbitol) aerosol, BID 2 4 IT Bleomycin 5 ml ofVehicle 0.025 U in 50 μl (0.625% poloxamer saline 188/10% sorbitol)aerosol, BID 3 5 IT Bleomycin 5 ml of 25 mg/ml 0.025 U in 50 μltegavivint aerosol, saline BID

Murine model of pulmonary fibrosis was induced by intratracheally (IT)injected bleomycin (APP Pharmaceuticals, Schaumburg, Ill.). One dose of0.025 U bleomycin dissolved in 50 microliters of Saline 0.9%, or PBS ascontrol was administered to each animal on day 0.

Tegavivint nanosuspension was applied to Group 3 in the form ofaerosols, through the method of whole body exposure. The mice wereplaced inside a plastic box. This box was sealed and connected by one ofits sides to the outlet of the nebulizer device, and on the other sideto a system of closed water. The whole procedure was carried out insidethe fume hood of the animal room. In each treatment session, 5 ml of 25mg/ml Tegavivint (125 mg) was nebulized over 15 min to each group of 4-5mice in the chamber. To increase exposure of the mice to the aerosol,Tegavivint that precipitated in the aerosol chamber was collected with asyringe and re-nebulized a second and a third time. Mice were nebulizedtwice a day between day 5 and day 21 after administration of bleomycin.Group 1 and 2 received nebulized vehicle 5 ml in the same manner.

The body weight of animals was recorded on Days 0, 5, 8, 12, 16, 19, and21.

Measurements of lung mechanics were performed on Day 21 as previouslydescribed (Morales-Nebreda L, et al. AJRCMB 2015) on Day 21 using aFlexiVent mouse ventilator (Scireq, Montreal, PQ, Canada) according tothe protocols established by Scireq. A standard ventilation history foreach mouse was obtained with three total lung capacity maneuvers beforethe forced oscillation and quasistatic pressure-volume curve protocolsthat were used to calculate airway resistance, dynamic and quasistatictissue compliance, and elastance.

On Day 21 all animals were sacrificed and lungs were harvested. Totallung collagen content was evaluated using the Hydroxyproline Assay aspreviously described (Morales-Nebreda L, et al. AJRCMB 2015). In brief,mouse lungs were harvested and suspended in 1 ml of 0.5 M acetic acidand then homogenized, first with a tissue homogenizer (60 s on ice) andthen using 15 strokes in a Dounce homogenizer (on ice). The resultinghomogenate was spun (12,000×g) for 10 minutes, and the supernatant wasused for subsequent analyses. Collagen standards were prepared in 0.5 Macetic acid using rat tail collagen (Sigma-Aldrich, St. Louis, Mo.).Picrosirius red dye was prepared by mixing 0.2 g of Sirius red F3B(Sigma-Aldrich) with 200 ml of water; 1 ml of the Picrosirius red dyewas added to 100 μl of the collagen standard or the lung homogenates andthen mixed continuously at room temperature on an orbital shaker for 30minutes. The precipitated collagen was then pelleted and washed oncewith 0.5 M acetic acid (12,000×g for 15 min each). The resulting pelletwas resuspended in 1 ml of 0.5 M NaOH and Sirius red staining wasquantified spectrophotometrically (540 nm) using a colorimetric platereader (Bio-Rad, Hercules, Calif.).

Results

Group 2 showed statistically significant body weight reduction afterbleomycin treatment, which is one of the indicators of IPF induction. Incontrast, inhaled tegavivint treatment in Group 3 reversed the bodyweight loss caused by the bleomycin induced lung injury.

Change in body weight (%) Animal # Group 1 Group 2 Group 3 1 3.24 −10.85−2.83 2 9.13 −1.25 5.2 3 9.85 −4.67 7.3 4 12 −2.62 7.3 5 7.9

Further, bleomycin induced decreased lung compliance in Group 2, whichindicates the induction of fibrosis. Inhaled tegavivint treatment afterbleomycin injury in Group 3 reversed the compliance values to near thoseof the sham-treated controls in Group 1.

Compliance (ml/cm H₂0) Animal # Group 1 Group 2 Group 3 1 0.0763370.044172 0.055153 2 0.068275 0.042324 0.056036 3 0.058057 0.0486670.067618 4 0.07324 0.042422 0.054295 5 0.056101

Further, the total lung collagen content as measured by theHydroxyproline assay showed marked increase in Group 2, indicatingactive fibrosis after bleomycin injury; in contrast, inhaled tegavivinttreatment after bleomycin injury in Group 3 reversed this change and thecollagen levels are closed to sham-treated controls in Group 1.

Hydroxyproline (μg/half lung) Animal # Group 1 Group 2 Group 3 1 51.29675.632 70.016 2 36.32 85.824 39.44 3 44.432 68.768 45.68 4 37.568 77.50458.784 5 64.296

What is claimed is:
 1. A method of treating and/or preventing a fibroticdisease, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, one of R₇ and R₈ is hydrogen, and R₁, R₂, R₃, and R₄ areeach independently selected from H, alkyl, heteroalkyl, cycloalkyl,arylcycloalkyl, aryl, heteroaryl, heterocycloalkyl, and each of NR₁R₂and NR₃R₄ can independently combine to form a heterocycloalkyl, andwherein said alkyl, heteroalkyl, cycloalkyl, arylcycloalkyl, aryl,heteroaryl, or heterocycloalkyl may be optionally substituted, or apharmaceutically acceptable salt, ester, amide, stereoisomer, geometricisomer or prodrug thereof.
 2. The method of claim 1, wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring.
 3. The method of claim 1, wherein the compoundof formula I has the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer,geometric isomer or prodrug thereof.
 4. The method of claim 1, whereinthe fibrotic disease is selected from the group consisting of pulmonaryfibrosis, Dupuytren's contracture, scleroderma, systemic sclerosis,scleroderma-like disorders, sine scleroderma, liver cirrhosis,interstitial pulmonary fibrosis, keloids, chronic kidney disease,chronic graft rejection, and other scarring/wound healing abnormalities,post-operative adhesions, reactive fibrosis.
 5. The method of claim 4,wherein said disorder is pulmonary fibrosis.
 6. The method of claim 4,wherein said disorder is Dupuytren's contracture.
 7. The method of claim4, wherein said disorder is keloids.
 8. A method of treating pulmonaryfibrosis comprising intranasally administering the compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or geometric isomer thereof, to a patient in needthereof.
 9. The method of claim 8, wherein the compound of formula I hasthe following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer,geometric isomer or prodrug thereof.
 10. The method of claim 1, whereinsaid administering is done through one or more of intravenous,parenteral, oral, inhalation (including aerosolized delivery), buccal,intranasal, rectal, intra-lesional intraperitoneal, intradermal,transdermal, subcutaneous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreous injection, andtopical application methods.